Mesenchymal stem cells are stem cells that can be isolated from a variety of tissues such as bone marrow, adipose tissue, dermis/skin, etc. These cells are the subject of intense scientific research and scrutiny and are thought to represent a cornerstone for potentially revolutionary paradigms of regenerative therapies of the future.
Mesenchymal stem cells in general, and adipose stem cells in particular, hold great promise for future clinical therapies which enhance the body's natural ability to heal itself. One hurdle common to the use of these potential therapies is the current practice of using fetal bovine serum or other animal sera in the culture media of cells intended for use in humans. The undefined and variable nature of animal sera, as well as the associated risk of introducing xenobiotic pathogens and triggering severe allergic responses in some subjects, presents a technical problem presently unresolved in the field.
In recent years, the identification of mesenchymal stem cells, chiefly obtained from bone marrow, has led to advances in tissue regrowth and differentiation. Such cells are pluripotent cells found in bone marrow and periosteum, and they are capable of differentiating into various mesenchymal or connective tissues. For example, such bone-marrow derived stem cells can be induced to develop into myocytes upon exposure to agents such as 5-azacytidine (Wakitani et al., Muscle Nerve, 18 (12), 1417-26 (1995)). It has been suggested that such cells are useful for repair of tissues such as cartilage, fat, and bone (see, e.g., U.S. Pat. Nos. 5,908,784, 5,906,934, 5,827,740, 5,827,735), and that they also have applications through genetic modification (see, e.g., U.S. Pat. No. 5,591,625). While the identification of such cells has led to advances in tissue regrowth and differentiation, the use of such cells is hampered by several technical hurdles. One drawback to the use of such cells is that they are very rare (representing as few as 1/2,000,000 cells), making any process for obtaining and isolating them difficult and costly. Of course, bone marrow harvest is universally painful to the donor. Moreover, such cells are difficult to culture without inducing differentiation, unless specifically screened sera lots are used, adding further cost and labor to the use of such stem cells.
The presence of adult multipotent “stem” cells has been demonstrated in a large number of tissues, for example the bone marrow, blood, liver, muscle, the nervous system, and in adipose tissue. Adult “stem” cells, which in theory are capable of infinite self-renewal, have great cell plasticity, i.e., the ability to differentiate into tissues other than those for which it was believed they were destined. The properties of said cells, which are similar to those of embryonic stem cells (ES), open up considerable therapeutic perspectives especially as their use does not pose the problems of compatibility and ethics, encountered with ES cells.
Adipose tissue plays an important and overlooked role in the normal development and physiology of humans and other mammalian species. Many different kinds of fat exist. The most common type is white adipose tissue, located under the skin (subcutaneous fat), within the abdominal cavity (visceral fat) and around the reproductive organs (gonadal fat). Less common in the adult human is brown adipose tissue, which plays an important role in generating heat during the neonatal period; this type of fat is located between the shoulder blades (interscapular), around the major vessels and heart (periaortic and pericardial), and above the kidney (suprarenal).
As women mature, they develop increased amounts of mammary adipose tissue. The mammary fat pad serves as an energy source during periods of lactation. Indeed, reproductive capacity and maturation are closely linked to the adipose tissue stores of the individual. Puberty in women and men correlates closely with the production and release of leptin, an adipose tissue derived hormone, and to body fat composition. Other adipose tissue sites play a structural role in the body. For example, the mechanical fat pads in the soles of the feet provide a cushion against the impact of walking. Loss of this fat depot leads to progressive musculoskeletal damage and impaired mobility. Bone marrow fat cells are present in bone marrow to provide energy to developing blood cells within the marrow.
Bone marrow adipocytes are different than adipocytes present in adipose tissue, differing in morphology, physiology, biochemistry as well as their response to various stimulators such as insulin. Adipocytes present in bone marrow stroma may function to: 1) regulate the volume of hemodynamically active marrow; 2) serve as a reservoir for lipids needed in marrow cell proliferation, and 3) may be developmentally related to other cell lineages such as osteoblasts. White adipose tissue (i.e. body fat) in contrast, is involved in lipid metabolism and energy homeostasis (Gimble, “The Function of Adipocytes in the Bone Marrow Stroma”, The New Biologist 2(4), 1990, pp. 304-312).
The vast majority of research related to various stem cell populations has centered on their behavior and therapeutic potential as adherent cell cultures and/or single cell suspensions that are either mixed in nature, or clonally derived. However, a consensus is evolving, supported by promising evidence, that stem cells most likely exist in vivo within the context of a supportive niche, or microenvironment.
As reviewed in several recent papers, emerging data suggest that “it is the combination of the intrinsic characteristics of stem cells and their microenvironment that shapes their properties and defines their potential” (Fuchs et al., Cell, 116:769-778, 2004). In essence, the specific cellular environment, or niche, is composed of a diverse, heterogeneous collection of cells (in addition to, or including the stem cell constituents) that create/provide a milieu of soluble and matrix factors. These factors help to direct and control the homeostasis of the stem cell reservoir, including cell growth, differentiation, and renewal (Kindler, J. Leukocyte Biol., 78:836-844, 2005; Fuchs et al., Cell, 116:769-778, 2004). And while it is currently thought that the majority of stem cells are dormant/quiescent in the G0 phase of the cell cycle when a tissue/niche is in equilibrium, it is also believed that loss of, or damage to a tissue/niche provides a powerful stimulus to the stem cell reservoir to re-establish equilibrium (i.e., repair; regenerate) by renewal (expansion) and/or differentiation. This capacity likely involves asymmetric cell division and possibly some degree of dedifferentiation, all of which is thought to be governed by the niche micromilieu.
Given the above background, it becomes clear that the ‘creation’ of ex vivo stem cell niche models would be highly useful and valuable for the study of stem cell biology, as well as for potential therapeutic applications. Researchers have described and characterized in vitro ‘niches’ for embryonic stem cells (embryoid bodies) and neural stem cells (neurospheres)—which both involve suspension (i.e., non-adherent) culture of said cells in multicellular aggregates. However, no such ‘system’ has been described for mesenchymal stem/stromal cells, particularly adipose-derived cells. Such cells are traditionally cultured as adherent monolayers, and this is likely due to the difficulty in culturing these cells in suspension, as they are extremely adherent, even to surfaces that are supposedly unfavorable to cell culture/adherence.
There is a long felt need in the art for methods to prepare, characterize, and manipulate multicellular aggregates of mesenchymal stem cells, particularly adipose tissue-derived cells, and to provide for alternative, and/or enhanced regenerative technologies. The present invention satisfies this need.